Influence of new calpain inhibitors KR-185 and KR-161 on some biochemical parameters in cancer and 3T3-L1 preadipocytes

Several studies have notably highlighted the involvement of calpains in the processes essential for cancer development and cell cycle progression, including; cell transformation, migration and tumor invasion, angiogenesis, apoptosis/survival. As a result, calpains are considered by several researchers as potential anti-cancer targets. So many calpain inhibitors have been synthesized with or without specificity for different isoforms of calpain. In this study, we investigated the effect of calpain inhibitors KR-185 and KR-161 on some biochemical parameters. These parameters are cell viability, calpain activity, antioxidant potentials (GSH assay) and Caspase-9 expression levels, which are essential for cancer development in humans. In this study, A549 lung cancer cells, HeLa cells, BT549 breast cancer cells and 3T3-L1 preadipocytes were exposed to either the same or different doses of the newly synthesized calpain inhibitors KR-185 and KR-161 after establishing a safe and working dose of 5, 10, 15, 20 and 25 μM or 0, 10, 20 30, 50 and 70 μM depending on the cell line. The effect of both calpain inhibitors on cytotoxicity was minimal even at high concentration. In comparison, the results obtained revealed that KR-161 are more specific than KR-185 for a wider range of calpain in cancer and fat cells. Reduction in calpain activity was observed in all cell lines when treated with KR-161 for 24 hours unlike KR-185 which was specific to only A549 lung cancer cells and BT549 breast cancer cells. Both calpain inhibitors were observed to reduce the endogenous glutathione levels of almost all cell lines but not below the normal physiological level (10 μM). KR-185 decreased the expression of caspase-9 in A549 lung cancer cells, HeLa cells and BT549 breast cancer cells. Its effect on the expression of caspase-9 in 3T3-L1 preadipocytes could not be assayed for due to the specificity of the kit for human caspase-9 not cells from mice

Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites.

Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia. This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII